D-FRUCTOSE and D-GLUCOSE

Transcription

1 DFRUCTOSE and DGLUCOSE (Liquid Stable Format) ASSAY PROCEDURE KFRGLQR 02/17 (1100 AutoAnalyser Assays of each per Kit) or (1100 Microplate Assays of each per Kit) Megazyme 2017

2 INTRODUCTION: DGlucose and Dfructose are found in most plant products. In foods, they are present in significant quantities in honey, wine and beer, and a range of solid foodstuffs such as bread and pastries, chocolate and candies. In the wine industry, the sum of Dglucose and Dfructose, termed total residual sugars, is a key parameter, as this represents the amount of sugar that is available to the yeast for the conversion into ethanol. Total residual sugar levels are monitored throughout fermentation and, after fermentation is complete, are adjusted to achieve the desired taste profile. For the vast majority of measurements taken during the wine making process it is unnecessary to differentiate between the Dglucose and Dfructose, allowing them to be quantified simultaneously. This kit (KFRGLQR) is suitable for the specific measurement of Dglucose and Dfructose in wines, beverages, foodstuffs and other materials. The procedure formats provided in this booklet allow for the individual or simultaneous measurement of Dglucose and Dfructose. PRINCIPLE: DGlucose and Dfructose are phosphorylated by the enzyme hexokinase (HK) and adenosine5 triphosphate (ATP) to glucose 6phosphate (G6P) and fructose6phosphate (F6P) with the simultaneous formation of adenosine5 diphosphate (ADP) (1), (2). (HK) (1) DGlucose + ATP G6P + ADP (HK) (2) DFructose + ATP F6P + ADP In the presence of the enzyme glucose6phosphate dehydrogenase (G6PDH), G6P is oxidised by nicotinamideadenine dinucleotide phosphate (NADP + ) to gluconate6phosphate with the formation of reduced nicotinamideadenine dinucleotide phosphate (NADPH) (3). (G6PDH) (3) G6P + NADP + gluconate6phosphate + NADPH + H + The amount of NADPH formed in this reaction is stoichiometric with the amount of Dglucose. It is the NADPH which is measured by the increase in absorbance at 340 nm. 1

3 On completion of reaction (3), F6P is converted to G6P by phosphoglucose isomerase (PGI) (4). (PGI) (4) F6P G6P The G6P formed reacts in turn with NADP + forming gluconate6 phosphate and NADPH, leading to a further rise in absorbance that is stoichiometric with the amount of Dfructose. SAFETY: The general safety measures that apply to all chemical substances should be adhered to. For more information regarding the safe usage and handling of this product please refer to the associated SDS that is available from the Megazyme website. KITS: Kits suitable for performing 1100 assays of each test in autoanalyser format or microplate format are available from Megazyme. The kits contain the full assay method plus: Bottle 1: Bottle 2: Bottle 3: Bottle 4: Reagent 1 (44 ml) Contains sodium azide (0.08% w/v) as a preservative. Ready to use. Stable for > 2 years at 4 C. Reagent 2 (22 ml) Contains sodium azide (0.05% w/v) as a preservative. Ready to use. Stable for > 2 years at 4 C. Reagent 3 (22 ml) Contains sodium azide (0.05% w/v) as a preservative. Ready to use. Stable for > 2 years at 4 C. DGlucose plus DFructose Standard (2 ml) (3 g/l of each sugar). Ready to use. Stable for > 2 years at room temperature. 2

4 AUTOANALYSER ASSAY PROCEDURES: NOTES: 1. To obtain individual measurements of Dglucose and Dfructose use AutoAnalyser Format A (page 3). Alternatively, to obtain a single combined measurement of Dglucose and Dfructose (e.g. total residual sugars) use AutoAnalyser Format B (page 4). 2. For each batch of samples that is applied to the determination of Dglucose or Dfructose either a single point standard or a calibration curve must be performed concurrently using the same batch of reagents. A. AUTOANALYSER FORMAT: D Glucose & D Fructose Wavelength: 340 nm Calculation: Endpoint Temperature: ~ 25 C or 37 C Reaction: Absorbance increase Final volume: ml (for Dglucose) ml (for Dfructose) Linearity: µg of Dglucose plus Dfructose per cuvette (in sample volume) (up to 6,000 mg/l of original sample) Pipette into cuvettes Sample Standard reagent 1 distilled water sample standard CALCULATION FORMULA: DGlucose A 2 (A 1 x 203/223) DFructose A 3 (A 2 x 223/243) 3 Mix, read the absorbances of the solutions (A 1 ) after 3 min and start the reactions by addition of: reagent 2 Mix and read the absorbances of the solutions (A 2 ) after 5 min. Then add: reagent 3 Mix and read the absorbances of the solutions (A 3 ) after 10 min.

5 B. AUTOANALYSER FORMAT: Total Residual Sugars Wavelength: 340 nm Calculation: Endpoint Temperature: ~ 25 C or 37 C Reaction: Absorbance increase Final volume: ml Linearity: µg of Dglucose plus Dfructose per cuvette (in sample volume) (up to 6,000 mg/l of original sample) Pipette into cuvettes Sample Standard reagent 1 distilled water sample standard Mix, read the absorbances of the solutions (A 1 ) after 3 min and start the reactions by addition of: reagent 2 reagent 3 Mix and read the absorbances of the solutions (A total ) after 10 min. CALCULATION FORMULA: Total Residual Sugars A total (A 1 x 203/243) 4

6 MICROPLATE ASSAY PROCEDURES: NOTES: 1. To obtain individual measurements of Dglucose and Dfructose use Microplate Format A (page 5). Alternatively, to obtain a single combined measurement of Dglucose and Dfructose (e.g. total residual sugars) use Microplate Format B (page 6). 2. For each batch of samples that is applied to the determination of Dglucose or Dfructose either a single point standard or a calibration curve must be performed concurrently using the same batch of reagents. A. MICROPLATE FORMAT: DGlucose & DFructose Wavelength: 340 nm Microplate: 96well (e.g. clear flatbottomed, glass or plastic) Temperature: ~ 25 C or 37 C Final volume: ml (for Dglucose) ml (for Dfructose) Linearity: µg of Dglucose plus Dfructose per cuvette (in sample volume) (up to 6,000 mg/l of original sample) Pipette into well Sample Standard Blank reagent 1 distilled water sample standard ml Mix* and read absorbances of the solutions (A 1 ) after exactly 3 min. Then add: reagent 2 Mix* and read the absorbances of the solutions (A 2 ) at the end of the reaction (approx. 5 min). If the reaction has not stopped after 5 min, continue to read the absorbances at 2 min intervals until the absorbances remain the same over 2 min. Then add: reagent 3 Mix* and read the absorbances of the solutions (A 3 ) at the end of the reaction (approx. 10 min). If the reaction has not stopped after 10 min, continue to read the absorbances at 2 min intervals until the absorbances remain the same over 2 min. * for example using microplate shaker, shake function on a microplate reader or repeated aspiration using a pipettor. 5

7 B. MICROPLATE FORMAT: Total Residual Sugars Wavelength: 340 nm Microplate: 96well (e.g. clear flatbottomed, glass or plastic) Temperature: ~ 25 C or 37 C Final volume: ml Linearity: µg of Dglucose plus Dfructose per cuvette (in sample volume) (up to 6,000 mg/l of original sample) Pipette into well Sample Standard Blank reagent 1 distilled water sample standard ml Mix* and read absorbances of the solutions (A 1 ) after exactly 3 min. Then add: reagent 2 reagent 3 Mix* and read the absorbances of the solutions (A total ) at the end of the reaction (approx. 10 min). If the reaction has not stopped after 10 min, continue to read the absorbances at 2 min intervals until the absorbances remain the same over 2 min. * for example using microplate shaker, shake function on a microplate reader or repeated aspiration using a pipettor. 6

8 Bray Business Park, Bray, Co. Wicklow, A98 YV29, IRELAND. Telephone: (353.1) Facsimile: (353.1) Internet: WITHOUT GUARANTEE The information contained in this booklet is, to the best of our knowledge, true and accurate, but since the conditions of use are beyond our control, no warranty is given or is implied in respect of any recommendation or suggestions which may be made or that any use will not infringe any patents. 7